Field of the Invention
The present invention relates to a method for the production of recombinant expression vectors, a kit adapted to carry out the method, a vector used in connection with the method, a cell containing said vector, and the use of the vector.
Related Prior Art
A central subject matter of molecular cloning is the production of recombinant nucleic acid molecules. For this purpose, a desired nucleic acid molecule, usually a DNA fragment or a gene, is integrated in a so-called vector or a “gene shuttle”, such as for example a plasmid or a viral vector. An object of cloning is to amplify the integrated DNA fragment or gene to examine its properties or to further use them in follow-up processes. After a multiplication the vector can be isolated and a multiple of the initially used DNA fragment can be gained. Alternatively, cells into which a DNA fragment was introduced via a vector by means of cloning, can express a gene product encoded by the DNA fragment, e.g. a protein or peptide. Such a vector is also referred to as expression vector.
Standardized experiments for the cloning of any DNA fragment essentially comprise the following seven steps: (1) selection of the host organism and the cloning vector, (2) production of the vector, (3) production of the DNA to be cloned, (4) generation of recombinant DNA, (5) introduction of the recombinant DNA into the host organism, (6) selection of organisms containing the recombinant DNA, (7) screening for clones with the desired cloned DNA and/or the desired biological properties.
One of the time-limiting factors in the production of recombinant (expression) vectors is the selection of such clones which express the introduced gene product. One of the frequently used methods is the so-called blue white selection. The objective of the blue white selection is the identification of cells which express the desired introduced gene. As a rule, for the blue white selection specific plasmids are used as vectors, which contain at the position for the insertion of the “gene of interest” into the plasmid at the so-called ‘multiple cloning site’ the gene for a β-galactosidase, the so-called lacZ gene. The gene for the galactosidase is used as reporter gene. By the introduction of the gene of interest into the multiple cloning site the galactosidase is inactivated. By this, after a transformation of the plasmids the transgenic organisms, in contrast to the nontransgenic organisms, do not contain a functional galactosidase. The galactosidase can cleave the yellow dye ‘X-gal’ into a blue dye and galactose. When using X-gal culture medium about half an hour after the induction a blue dye is produced by the galactosidase contained in the cells. However, in the transgenic cells the galactosidase is inactivated in which case they remain undyed and can be isolated with reference to their lack of dye.
This selection method is very time-consuming and labor-intensive. It is not unusual that in particular for the production of recombinant virus based expression vectors up to 6 to 12 month are required which is, for example, unfavorable for the production of seasonally required or individualized vaccines.
Other methods for the production or selection of recombinant vectors currently used in the state of the art are likewise time-consuming and labor-intensive.